Recently, type I cells have been implicated as potential mechanosensors for stimulating surfactant secretion by type II cells by intercellular transmission of transient increases in cytosolic calcium (calcium waves). There are two major pathways for this: gap junctional intercellular communication and paracrine stimulation of purinergic receptors. We will use a combination of approaches to determine which intercellular signaling pathways regulate surfactant secretion. Using chemically distinct inhibitors with overlapping modes of action, we will determine whether disrupting different intercellular communication pathways inhibits secretion of metabolically labeled endogenous surfactant lipid (Aim 1). This will enable us to determine whether increased surfactant lipid in response to increases in ventilation parameters are due to signals propagated through gap junctions, purinergic receptors, or both. In Aim 2, co-cultures systems will be used to identify intercellular communication pathways used to transmit heterocellular calcium waves and to stimulate surfactant secretion. Secretion Surfactant producing cells used for these studies include adult primary type II cells. While adult type II cells express P2Y2 purinergic receptors, neonatal cells do not, providing the equivalent of a "knock-out" model for this receptor As partners for type II cells, we will use type II cells cultured for 6 days (Day 6 cells) as the best available model for type I-like cell in culture. We will also use other cells as partners for type II cells, including Hela transfectants with differing connexin expression. Preliminary data indicates that co-culture of adult type II cells with Day 6 cells stimulates surfactant secretion. The effect of inhibitors on calcium wave transmission and surfactant secretion will be examined in co-cultures. Day 6 cells transduced with adenovectors encoding dominant negative Cx43 constructs will also help elucidate roles for Cx43 in stimulating surfactant secretion. The effect of inhibitors on calcium wave transmission and surfactant secretion will be examined in co-cultures. Day 6 cells transduced with adenovectors encoding dominant negative Cx43 constructs will help elucidate roles for Cx43 in stimulating surfactant secretion. In Aim 3, we will define the purinergic receptor expression profile for primary alveolar epithelial cells with different phenotypes in culture and in situ. Finally, in Aim 4, we will use heterocellular co-cultures of alveolar epithelial cells cultured on deformable membrane substrate to determine whether mechanical distension induces intercellular signals transmitted to type II cells. By defining the molecular the molecular constituents of cell-cell communication important for regulating surfactant secretion, we hope to identify possible targets that are disrupted during acute lung injury or ventilator induced injury and that may exacerbate the extent of lung disease by causing misregulation of surfactant secretion.